Tronsmo A, Harman G E
Department of Horticultural Sciences, Cornell University, New York State Agricultural Experiment Station, Geneva 14456.
Anal Biochem. 1993 Jan;208(1):74-9. doi: 10.1006/abio.1993.1010.
Procedures are described for the direct assay of N-acetyl-beta-glucosaminidase (EC 3.2.1.30), chitobiosidase, and endochitinase (EC 3.2.1.14) after separation on starch or polyacrylamide electrophoresis gels. The enzymes were visualized as fluorescent bands by using an agarose overlay containing 4-methylumbelliferyl derivatives of N-acetyl-beta-D-glucosaminide, beta-D-N,N'-di-acetylchitobioside, or beta-D-N,N',N"-triacetylchitotriose for N-acetyl-beta-glucosaminidase, chitobiosidase, or endochitinase, respectively. For quantitative assay of N-acetyl-beta-glucosaminidase and chitobiosidase in solutions, a rapid technique using nitrophenyl-N-acetyl-beta-D-glucosaminide and nitrophenyl-beta-D-N,N'-diacetyl-chitobiose, respectively, was used. Endochitinase activity was quantitatively measured by determining the percentage reduction in turbidity of a reaction mixture that contained purified colloidal chitin. Trichoderma harzianum strain P1 was shown to produce three kinds of chitinolytic enzymes, and there were multiple forms of some of these.
本文描述了在淀粉或聚丙烯酰胺电泳凝胶上分离后直接测定N-乙酰-β-葡萄糖苷酶（EC 3.2.1.30）、壳二糖酶和内切几丁质酶（EC 3.2.1.14）的方法。通过使用含有N-乙酰-β-D-葡萄糖胺、β-D-N，N'-二乙酰壳二糖或β-D-N，N'，N''-三乙酰壳三糖的4-甲基伞形酮衍生物的琼脂糖覆盖物，分别将N-乙酰-β-葡萄糖苷酶、壳二糖酶或内切几丁质酶可视化为荧光带。对于溶液中N-乙酰-β-葡萄糖苷酶和壳二糖酶的定量测定，分别使用了一种快速技术，即分别使用对硝基苯基-N-乙酰-β-D-葡萄糖胺和对硝基苯基-β-D-N，N'-二乙酰壳二糖。通过测定含有纯化胶体几丁质的反应混合物浊度的降低百分比来定量测量内切几丁质酶活性。哈茨木霉P1菌株显示产生三种几丁质分解酶，并且其中一些有多种形式。
